Method for large-scale production of di(uridine 5&#39;)-tetraphosphate and salts thereof

ABSTRACT

The present invention provides new methods for the synthesis of the therapeutic dinucleotide, P 1 ,P 4 -di(uridine 5′-tetraphosphate), and demonstrates applicability to the production of large quantities. The methods of the present invention substantially reduce the time period required to synthesize diuridine tetraphosphate, preferably to three days or less. The novel tetrammonium and tetrasodium salts of P 1 ,P 4 -di(uridine 5′-tetraphosphate) (Formula I) prepared by these methods are stable, soluble, nontoxic, and easy to handle during manufacture.  
                 
 
     wherein:  
     X is Na, NH 4  or H, provided that all X groups are not H.

[0001] This application claims priority to U.S. Provisional Application60/054,147 filed Jul. 25, 1997.

TECHNICAL FIELD

[0002] This invention relates to methods for the production oftherapeutic dinucleotides including novel salts thereof. Morespecifically, it relates to methods for synthesis of P¹,P⁴-di(uridine5′-tetraphosphate), i.e., diuridine tetraphosphate (U₂P₄) which haveadvantages over prior art methods of manufacture.

BACKGROUND OF THE INVENTION

[0003] P¹,P⁴-Di(uridine 5 ′-tetraphosphate) is a dinucleotide of thefollowing structure:

[0004] wherein:

[0005] X is Na, NH₄ or H, provided that all X groups are not H.

[0006] The free acid of P¹,P⁴-di(uridine 5′-tetraphosphate), where X ishydrogen, has been previously described as uridine 5′-(pentahydrogentetraphosphate), P′″→5′-ester with uridine (CAS Registry Number:59985-21-6; C. Vallejo et al., Biochimica et Biophysica Acta 438, 305(1976) and H. Coste et al., J. Biol. Chem. 262, 12096 (1987)).

[0007] Different methods have been described for the synthesis of purinedinucleotides such as diadenosine tetraphosphate (A₂P₄) (E. Rappaport etal, Proc. Natl. Acad. Sci, 78, 838, (1981); A. Guranowski et al,Biochemistry, 27, 2959, (1988); C. Lobaton et al, Eur. J. Biochem., 50,495, 1975; K. Ng and L. Orgel, NucL. Acid Res., 15, 3573, (1987)).However, this has not been true for U₂P₄ which is a pyrimidinenucleotide. Although purine nucleotides and pyrimidine nucleotidesappear to be analogous, the methods used for purine nucleotide synthesisdo not necessarily work for pyrimidines such as uridine.

[0008] Diuridine tetraphosphate has been shown to have beneficialproperties in the treatment of various diseases, such as chronicobstructive pulmonary disease (COPD). For example, they have beendemonstrated to facilitate the clearance of mucous secretions from thelungs of a subject such as a mammal including humans in need oftreatment for various reasons, including cystic fibrosis, chronicbronchitis, asthma, bronchiectasis, post-operative mucous retention,pneumonia, primary ciliary dyskinesia (M. J. Stutts, III, et al, U.S.Pat. No. 5,635,160; PCT International Publication WO 96/40059) and theprevention and treatment of pneumonia in immobilized patients (K. M.Jacobus and H. J. Leighton, U.S. Pat. No. 5,763,447). Furthertherapeutic uses include treatment of sinusitis (PCT InternationalPublication WO 98/03177), otitis media (PCT International Publication WO97/29756), dry eye, retinal detachment, nasolacrimal duct obstruction,the treatment of female infertility and irritation due to vaginaldryness via increased mucus secretions and hydration of the epithelialsurface, and enhancing the performance of athletes.

[0009] U₂P₄ also has utility as a veterinary product in mammals such as,but not limited to, dogs, cats and horses.

[0010] Prior art methodology describes only one protocol for theproduction of diuridine tetraphosphate. This method is very timeconsuming, lasting over five days and producing only small amounts ofdiuridine tetraphosphate (C. Vallejo et al., Biochimica et BiophysicaActa 438, 305 (1976), Sillero et al., Eur J Biochem 76, 332 (1972)).According to this technique, diuridine tetraphosphate was synthesizedthrough a reaction of uridine 5′-monophosphomorpholidate (0.54 mmol)with the triethylamine salt of pyrophosphoric acid (0.35 mmol) in amedium of anhydrous pyridine (10 ml). After 5 days at 30° C., pyridinewas removed from the reaction mixture by evaporation, and the residueresuspended in glass-distilled water (8 mL), the suspension applied to aDEAE-cellulose column (37.5×2.6 cm) and fractionated with 3.2 L of alinear gradient (0.06-0.25 M) of ammonium bicarbonate, pH 8.6. The peakeluting between 0.17-0.19 M ammonium bicarbonate was partiallycharacterized as U₂P₄ by the following criteria: insensitivity toalkaline phosphatase, phosphorus to base ratio and analysis of theproducts of hydrolysis (UTP+UMP), after treatment with phosphodiesteraseI, by electrophoresis in citrate buffer, pH 5.0. No yield orspectroscopic data were given. Thus, the prior art procedure for thesynthesis of diuridine tetraphosphate is lengthy and produced only smallamounts of only partially characterized diuridine tetraphosphate. Thepresent invention focuses on methods to produce this medically usefulcompound which may be more efficiently and conveniently carried out, andwhich may be applied to the large-scale production of diuridinetetraphosphate and salts thereof.

SUMMARY OF THE INVENTION

[0011] The present invention provides new methods for the synthesis ofthe therapeutic dinucleotide, P¹,P⁴-di(uridine 5′-tetraphosphate)(Formula I), and demonstrates applicability to the production of largequantities. The methods of the present invention substantially reducethe time required to synthesize diuridine tetraphosphate, preferably tothree days or less. The novel ammonium and sodium salts ofP¹,P⁴-di(uridine 5′-tetraphosphate) prepared by these methods arestable, soluble, nontoxic, and easy to handle during manufacture. Thetetraammonium salt is preferred; the tetrasodium salt is most preferred.

[0012] wherein:

[0013] X is Na, NH₄ or H, provided that all X groups are not H.

[0014] The method of synthesizing compounds of Formula I, andpharmaceutically acceptable salts thereof, is carried out generally bythe following steps: 1) dissolving uridine or uridine nucleotidecompounds of Formulas IIa-d in a polar, aprotic organic solvent and ahydrophobic amine; 2) phosphorylating with a phosphorylating agent ofFormulas IVa-b and/or activating with an activating agent of FormulasIIIa-c; and 3) purifying by ion exchange chromatography.

[0015] Another aspect of the present invention are methods of treatingvarious disease states, including, but not limited to: chronicobstructive pulmonary diseases, sinusitis, otitis media, nasolacrimalduct obstruction, dry eye disease, retinal detachment, pneumonia, andfemale infertility or irritation caused by vaginal dryness.

[0016] Another aspect of the present invention is a pharmaceuticalcomposition comprising a compound of Formula I together with apharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention provides new methods for the synthesis ofthe therapeutic dinucleotide, P¹,P⁴-di(uridine 5′-tetraphosphate), anddemonstrates applicability to the production of large quantities. Themethods of the present invention substantially reduce the time periodrequired to synthesize P¹,P⁴-di(uridine 5′-tetraphosphate), preferablyto three days or less. The ammonium and sodium salts of P¹,P⁴-di(uridine5′-tetraphosphate) (Formula I) prepared by these methods are stable,soluble, nontoxic, and easy to handle during manufacture.

[0018] The present invention further provides compounds of Formula I:

[0019] wherein:

[0020] X is Na, NH₄ or H, provided that all X groups are not H.

[0021] The sodium and ammonium salts of P¹,P⁴-di(uridine5′-tetraphosphate) have many advantages. The sodium and ammonium saltsprovide good long-term stability profiles compared to those of divalentcations (e.g. Ca²⁺, Mg²⁺, Mn²⁺) which catalyze hydrolysis of phosphateesters. The tetrasodium salt of P¹,P⁴-di(uridine 5′-tetraphosphate) isnon-irritating to the lung and eyes. Other cations may be irritating tothe lungs, eyes, and other mucosal epithelia, or are otherwise not welltolerated by the human body. These inorganic sodium and ammonium saltsimpart excellent water solubility compared to hydrophobic amine saltssuch as tri- and tetrabutylammonium, and similar salts. High watersolubility is an important advantage for flexibility in pharmaceuticalformulations of varying concentration. The tetrammonium and tetrasodiumsalts of P¹,P⁴-di(uridine 5′-tetraphosphate) are also advantageous inthat they are readily purified by aqueous ion chromatography in which noorganic solvents are used. In addition, these salts are easily handledas fluffy, white solids, compared to an oil or gum as with some aminesalts.

[0022] The tetrasodium salt is preferred.

[0023] The compounds of Formula I may be used to facilitate theclearance of mucous secretions from the lungs of a subject such as amammal including humans in need of treatment for various reasons,including cystic fibrosis, chronic bronchitis, asthma, bronchiectasis,post-operative mucous retention, pneumonia, primary ciliary dyskinesia(M. J. Stutts, III, et al, U.S. Pat. No. 5,635,160; PCT InternationalPublication WO 96/40059) and the prevention and treatment of pneumoniain immobilized patients (K. M. Jacobus and H. J. Leighton, U.S. Pat. No.5,763,447). Further therapeutic uses include treatment of sinusitis (PCTInternational Publication WO 98/03177), otitis media (PCT InternationalPublication WO 97/29756), dry eye, retinal detachment, nasolacrimal ductobstruction, the treatment of female infertility and irritation due tovaginal dryness via increased mucus secretions and hydration of theepithelial surface, and enhancing the performance of athletes.

[0024] The compounds of Formula I may be administered orally, topically,parenterally, by inhalation or spray, intra-operatively, rectally, orvaginally in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles. The termtopically as used herein includes patches, gels, creams, ointments,suppositiories, pessaries, or nose, ear or eye drops. The termparenteral as used herein includes subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques. Inaddition, there is provided a pharmaceutical formulation comprising acompound of general Formula I and a pharmaceutically acceptable carrier.One or more compounds of general Formula I may be present in associationwith one or more non-toxic pharmaceutically acceptable carriers ordiluents or adjuvants and, if desired, other active ingredients. Onesuch carrier would be sugars, where the compounds may be intimatelyincorporated in the matrix through glassification or simply admixed withthe carrier (e.g., lactose, sucrose, trehalose, mannitol) or otheracceptable excipients for lung or airway delivery.

[0025] One or more compounds of general Formula I may be administeredseparately or together, or separately or together with: mucolytics suchas DNAse (Pulmozyme®)) or acetylcysteine, antibiotics, including but notlimited to inhaled Tobramycin®; non-steroidal anti-inflammatories,antivirals, vaccines, decongestants and corticosteroids.

[0026] The pharmaceutical compositions containing compounds of generalFormula I may be in a form suitable for oral use, for example, astablets, caplets, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsion, hard or soft capsules, or syrups orelixirs. Compositions intended for oral use may be prepared according toany method known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets contain theactive ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for the manufacture of tablets.These excipients may be, for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example, starch, gelatin oracacia; and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed.

[0027] Formulations for oral use may also be presented as hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin, oras soft gelatin capsules wherein the active ingredients is mixed withwater or an oil medium, for example, peanut oil, liquid paraffin orolive oil.

[0028] Aqueous suspensions contain the active materials in admixturewith excipients suitable for the manufacture of aqueous suspensions.Such excipients are suspending agents, for example: sodiumcarboxymethylcellulose, methylcellulose and sodium alginate. Dispersingor wetting agents may be a naturally-occurring phosphatide orcondensation products of an allylene oxide with fatty acids, orcondensation products of ethylene oxide with long chain aliphaticalcohols, or condensation products of ethylene oxide with partial estersfrom fatty acids and a hexitol, or condensation products of ethyleneoxide with partial esters derived from fatty acids and hexitolanhydrides. Those skilled in the art will recognize the many specificexcipients and wetting agents encompassed by the general descriptionabove. The aqueous suspensions may also contain one or morepreservatives, for example, ethyl, or n-propyl p-hydroxybenzoate, one ormore coloring agents, one or more flavoring agents, and one or moresweetening agents, such as sucrose or saccharin.

[0029] Dispersible powders and granules suitable for preparation of anaqueous suspension by the addition of water provide the activeingredients in admixture with a dispersing or wetting agent, suspendingagent and one or more preservatives. Suitable dispersing or wettingagents and suspending agents are exemplified by those already mentionedabove. Additional excipients, for example, sweetening, flavoring, andcoloring agents, may also be present.

[0030] Compounds of Formula I may be administered parenterally in asterile medium. The drug, depending on the vehicle and concentrationused, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anaesthetics, preservatives andbuffering agents can be dissolved in the vehicle. The sterile injectablepreparation may be a sterile injectable solution or suspension in anon-toxic parentally acceptable diluent or solvent. Among the acceptablevehicles and solvents that may be employed are sterile water, salinesolution, or Ringer's solution. The compounds of general Formula I mayalso be administered in the form of suppositories for ear, rectal orvaginal administration of the drug. These compositions can be preparedby mixing the drug with a suitable non-irritating excipient which issolid at ordinary temperatures but liquid at the body temperature andwill therefore melt to release the drug. Such materials are cocoa butterand polyethylene glycols.

[0031] Solutions of compounds of Formula I may be administered byintra-operative installation at any site in the body.

[0032] Single dosage levels of the order of from about 1 to about 400mg, preferably in the range of 10 to 300 mg, and most preferably in therange of 25 to 250 mg, are useful in the treatment of theabove-indicated respiratory conditions. Single dosage levels of theorder of from about 0.0005 to about 5 mg, preferably in the range of0.001 to 3 mg and most preferably in the range of 0.025 to 1 mg, areuseful in the treatment of the above-indicated ophthalmic conditions.The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. It will beunderstood, however, that the specific dose level for any particularpatient will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,sex, diet, time of administration, route of administration, and rate ofexcretion, drug combination and the severity of the particular diseaseundergoing therapy.

[0033] The synthetic methods described below encompass several syntheticstrategies for producing P¹,P⁴-di(uridine 5′-tetraphosphate). Generally,all the methods use uridine or uridine nucleotide compounds from FormulaIIa-d as starting materials, which are dissolved in a polar, aproticorganic solvent (e.g. dimethylformamide, dimethylsulfoxide, dioxane,N-methylpyrrolidone, trimethylphosphate) and a hydrophobic amine (e.g.triethylamine, tributylamine, trioctylamine, 2,4,6-collidine,tetrabutylamrnonium, tri- and tetra-alkyl amines, heterocyclic amines).The product is obtained by phosphorylating with a phosphorylating agentfrom Formula IV (e.g. phosphorus oxychloride, pyrophosphate,pyrophosphorylchloride) or activating a phosphate group with anactivating agent from Formula III (e.g. carbonyldiimidazole, an alky oraryl carbodiimide, an alkyl or aryl phosphochloridate), respectively,with subsequent purification various means well known to those of skillin the art, including, but not limited to, ion chromatography (e.g. DEAESephadex, DEAE cellulose, Dowex 50, anion and cation exchange resins).

[0034] The pyrimidine β-D-ribofuranosyl starting materials uridine,uridine 5′-monophosphate (UMP), uridine 5′-diphosphate (UDP), anduridine 5′-triphosphate (UTP) are shown as free acids in Formulas IIa-dbelow, respectively. These materials are all commercially available inlarge quantity in various salt forms.

[0035] and salts thereof;

[0036] and salts thereof;

[0037] and salts thereof.

[0038] The activating agents carbodiimide, activated carbonyl, andactivated phosphorus compounds are shown in the general Formulas IIIa-cbelow, respectively.

[0039] wherein R¹ and R₂ are C₁-C₈ alkyl or cycloalkyl, C₁-C₈ optionallysubstituted alkyl or cycloalkyl (e.g. hydroxy and amino groups); aryl oroptionally substituted aryl (e.g. hydroxy and amino groups). Preferredcompounds of Formula IIIa are dicyclohexylcarbodiimide and1-(3-Dimethylarninopropyl)-3-ethylcarbodiimide hydrochloride.

[0040] wherein X is imidazole, tetrazole, and/or halogen. Preferredcompounds of Formula IIIb are carbonyldiimidazole andcarbonylditriazole.

[0041] wherein R¹ and R₂ are C₁-C₈ alkyl or cycloalkyl, C₁-C₈ optionallysubstituted alkyl, alkoxy or cycloalkyl (e.g. hydroxy and amino groups);aryl, alkoxy or optionally substituted aryl or alkoxy (e.g. hydroxy andamino groups) and/or halogen; and X is halogen. Preferred compounds ofFormula IIIc are diphenylphosphorochloridate, phenylphosphorodichloridate, phenylphosphonic dichloride anddiphenylphosphinic chloride.

[0042] The mono- and diphosphorylating agents are shown below in thegeneral formulas IVa-b.

[0043] wherein X is halogen. Preferred compound of Formula IVa isphosphorus oxychloride.

[0044] wherein X is oxygen, hydroxy, or halogen, and salts thereof.Preferred compounds of Formula IVb are pyrophosphoryl chloride andpyrophosphate.

[0045] Those having skill in the art will recognize that the presentinvention is not limited to the following examples and that the steps inthe following examples may be varied.

EXAMPLE 1 Method for the Production of Diuridine TetraphosphateTetrasodium Salt Using Uridine 5′-Diphosphate

[0046] Uridine 5′-diphosphate disodium salt (Yamasa, Choshi, Japan; 600grams) was dissolved in deionized water (5.4 L). The solution was passedthrough a Dowex 50W×4 H⁺ (Dow Chemical) column. The fractions containinguridine 5′-diphosphate were pooled and neutralized with tributylamine(Aldrich, St. Louis; 300 mL). The neutralized fractions wereconcentrated to an oil by using a rotary evaporator at a bathtemperature of 55-60° C. The oil was dissolved in dry dimethylformamide(Aldrich, 3 L) and then dried by concentrating to an oil using a rotaryevaporator (55-60° C. bath temperature). This step was repeated twice.The oil was again dissolved in dimethylformamide (3 L) and1,1-carbonyldiimidazole (Aldrich; 100 g) was added. The solution washeated at 50° C. for 2½ hours. An additional amount of activating agent(33 grams) was added and heating continued for a further 2½ hours. Thesolution was again concentrated to an oil on a rotary evaporator (bathtemperature at 55-60° C). The resulting oil was dissolved in deionizedwater to a conductivity equal to that of 0.2 M NH₄HCO₃. The solution wasthen loaded into a column of Sephadex DEAE-A25 (Pharmacia, Upsala,Sweden; pre-swollen in 1.0 M NaHCO₃ and washed with 2 column volumes ofdeionized H₂O). The column was eluted with the following solutions inthe following order: 60 L of 0.25 M NH₄HCO₃, 120 L of 0.275M NH₄HCO₃, 40L of 030 M NH₄HCO₃ and 40 L of 0.35 M NH₄HCO₃. The fractions havingsufficient amounts of pure diuridine tetraphosphate were pooled asdetermined by HPLC analysis and concentrated on a rotary evaporator(bath temperature at 55-60° C.). The resulting residue was dissolved indeionized water (1.5 L) and concentrated on a rotary evaporator. Thisstep was repeated 15 times or until excess of bicarbonate buffer wasremoved. The resulting oil was dissolved in a sufficient amount ofdeionized water to form a ca. 10% solution, the solution charged to aDowex 50W×4 Na⁺ (Dow) column and eluted with deionized water. Thefractions containing U₂P₄ were pooled and concentrated to a ca. 10-15%solution, which was lyophilized to yield U₂P₄ tetrasodium salt as awhite solid (150 g approximately 25% yield based on uridine5′-diphosphate).

Structure Elucidation of P¹,P⁴-di(uridine 5′-tetraphosphate) TetrasodiumSalt

[0047] Due to the lack of adequate spectroscopic data of nonadenylateddinucleotides in the literature, a full structure elucidation ofP¹,P⁴-di(uridine 5′-tetraphosphate), tetrasodium salt was performed byemploying modern analytical techniques. The molecular weight wasdetermined by mass spectrometry to be 878 [m/z 855, (M-Na⁺)⁻],confirming the molecular formula C₁₈H₂₂N₄O₂₃P₄•4Na. The exact massmeasured for C₁₈H₂₂N₄O₂₃P₄•3Na [(M-Na⁺)⁻: calculated 854.9318] was854.9268. The measured mass differed from the theoretical mass by 5.0milimass units (5.9 ppm) for a confidence level of 99.7%. Karl Fishermoisture analysis gave a value of 1.73% H₂O and further confirmation ofthe molecular formula was obtained from elemental analysis: calculatedfor Na=10.70, found 10.81%; C:P ratio calculated 1.74, found 1.80, basedon the molecular formula: C₁₈H₂₂N₄O₂₃P₄•4.2Na•1.1H₂O (FW=902.4 g/mol).The infrared spectrum showed a broad signal at 3422 cm⁻¹ and a signal at1702 cm⁻¹, indicating the presence of hydroxyl (O—H stretch) andcarbonyl (C=O stretch) functional groups. In addition, a phosphate P=Ostretch was observed at 1265 cm⁻¹. The UV spectrum in water displayed aλ_(max) of 262 nm with a molar absorptivity (ε) of 17,004. The specificrotation at 25° C. (c=1, H₂O) was determined by polarimetry to be−9.560°.

[0048] The NMR spectra are: ¹H NMR (D₂O, TMS) δ4.11 (m, 2H), 4.14 (m,1H), 4.25 (m, 1H), 4.27 (m, 1H), 5.84 (d, J=8.1 Hz, 1H), 5.86 (d, J=5.4Hz, 1H), 7.81 (d, J=8.1 Hz); ¹³C NMR (D₂O, TMS) δ65.1 (d, J=5.5 Hz),69.7, 73.5, 83.4 (d, J=9.4 Hz), 88.1, 102.8, 141.5, 152.9, 167.5; ³¹PNMR (D₂O, H₃PO₄ std) δ−22,32 (m),−10.75 (m). The ¹H coupled ³¹P spectrumshowed a broadening of the multiplet at δ−10.75 ppm due to theintroduction of ¹H coupling. This multiplet was therefore confirmed asP_(α). There was no effect of ¹H coupling on the multiplet at −22.23ppm, assigning this by default as P_(β). A Nuclear Overhauser Effect(NOE) was observed for H₆ to the H_(2′) and H_(3′) sugar protons.Because it is not possible for H₅ to show an NOE to the sugar protons,H₆ is confirmed. Additionally, N₁ substitution is confirmed, because nopyrimidine-sugar NOE is possible for an N₃ substituted structure.

[0049] Additional 2-dimensional NMR experiments were conducted to verifyconnectivity. HMQC shows connectivity for H₅ to C₅ and H₆ to C₆,confirming C₅ and C₆. COSY and NOE connectivity were observed for H₅ toH_(6′) verifying H₅. HMBC 3-bond connectivity was observed for: H₆ toC_(1′), C₆ to H_(1′), H_(1′), to C₂, H₆ to C₂. These data thus confirmH₁, C₂ and N₁ substitution. COSY connectivity of H_(1′) to H_(2′)confirms H_(2′) and HMQC connectivity of H_(1′) to C_(1′) and H_(2′) toC_(2′) confirms C_(1′) and C_(2′). Additionally, HMBC shows 2-bond Jconnectivity from H₅ to C₄, confirming C₄. A ¹³C DEPT spectrum withmult=1.5 shows the carbon at δ65.1 inverted relative to all othercarbons. This observation confirms that C_(5′) is a methylene. Thecoupling of ³¹P to carbons at δ65.1 and 83.4 confirms C_(5′) and C_(4′),because C_(4′) is the only coupled methyne. In addition, HMQC showsconnectivity for C_(5′) to H_(5′) and C_(4′) to H_(4′), confirmingH_(4′) and H_(5′). An NOE was observed for H_(1′) to H_(4′), H₆ toH_(2′) and H₆ to H_(3′), confirming the β anomer sugar configuration.

[0050] In conclusion, P¹,P⁴-di(uridine 5′-tetraphosphate), tetrasodiumsalt was synthesized on a 150 g scale in 25% yield from commerciallyavailable starting materials with a total reaction time of 5 hours. Thecrude product was efficiently purified by ion exchange chromatographyand the structure of the reaction product was unambiguously proven usingmass spectroscopic, NMR and other analytical techniques.

EXAMPLE 2 Method for the Production of Diuridine TetraphosphateTetrammonium Salt Using Uridine 5′-Monophosphate

[0051] Uridine 5′-monophosphate (Sigma, Milwaukee, 3.0 g, 9.26 mmol) wasdissolved in dry DMF (10 mL) and tributylamine (Aldrich, 2 mL). Thesolution was evaporated in vacuo at 40° C. to an oil. The residue wasdissolved in dry DMF (Aldrich, 8 mL) to form a solution.Carbonyldiimidazole (Aldrich, 1.65 g, 10.18 mmol) was added to thissolution. The reaction was heated at 50° C. for one hour. Uridine5′-triphosphate (Yamasa, 5.60 g, 10.18 mmol) prepared as the anhydroustributylanunonium salt in DMF (5 mL) and tributylamine (2 mL), asdescribed in Example 3 below, was added to the reaction solution. Themixture was allowed to stir at 50° C. for three days when the solutionwas evaporated in vacuo to an oil, redissolved in water (5 mL) andpurified by column (300×50 mm) chromatography (Sephadex DEAE-A25,40-120μ, Aldrich, pre-swollen in 1.0 M NaHCO₃ and washed with 2 columnvolumes of deionized H₂O (H₂O→0.3 M NH₄HCO₃ gradient). The purefractions were concentrated in vacuo at 35° C., and H₂O added andreevaporated 5 times to obtain diuridine tetraphosphate tetrammoniumsalt as a white solid (2.37 g, 30% yield): 92.11% pure by HPLC with thesame retention time as the standard. In addition, the tetrammonium saltwas analyzed by FABMS to give a mass of [C₁₈H₂₅N₄O₂₃P₄ (M—H⁺)³¹:calculated 788.9860] 788.9857, confirming a parent formula ofC₁₈H₂₆N₄O₂₃P₄ for the free acid].

EXAMPLE 3A Method for the Production of Diuridine Tetraphosphate UsingUridine 5′-Triphosphate (UTP)

[0052] A solution of uridine 5′-triphosphate (UTP) trisodium salt(ProBioSint, Varese, Italy; 5.86 g, 0.01 mol) in water (5 mL) was passedthrough a column of BioRad AG—MP 50 (Aldrich) strong cation exchangeresin in its tributylamine form (50 mL bed volume) and eluted withdistilled water (about 300 mL). To this solution was added tributylamine(Aldrich; 5 mL), and the suspension shaken until the pH of the aqueousfraction had risen to δ. The layers were separated and the aqueoussolution evaporated to small volume, then lyophilized overnight. Theresidue was dissolved in dry dimethylformamide (Aldrich; 20 mL) and thesolvent evaporated at 0.1 mmHg. The dried tributylamine salt was made upto 100 mL with anhydrous acetone to yield a stock solution (0.1 M inUTP). Dicyclohexylcarbodiimide (DCC) (Baker, Phillipsburg; 0.227 g, 1.2mmol) was added to an aliquot of the foregoing UTP solution (10 mL, 1.0mmol) and the solution stirred at room temperature for 30 minutes. Themixture was added to the triethylamine salt of uridine 5′-monophosphate(2.0 mmol, prepared by addition of triethylamine (0.5 mL) to a solutionof uridine 5′-monophosphate (UMP) (Sigma; 0.648 g in DMF), andevaporating to dryness). This suspension was then evaporated to dryness,the residue made up to 5.0 mL in dry DMF, and set aside at 40° C. for 24hours. The reaction mixture was separated by semipreparativeion-exchange chromatography (Hamilton PRP X-100 column), eluting with agradient of 0-1.0 M ammonium bicarbonate, 5 mL/min, 30 minutes. Thedinucleotide tetraphosphate eluted between 21 and 23 minutes; theproduct (76.7% yield based on UTP) was quantitated by comparison of itsultraviolet absorption at λ_(max) 263 nm with that of a standardsolution of P¹,P⁴-di(uridine 5′-tetraphosphate).

EXAMPLE 3B Method for the Production of Diuridine Tetraphosphate UsingUridine 5′-Triphosphate (UTP) and an Excess of Activating Agent

[0053] Conversion of UTP to P¹,P⁴-di(uridine 5′-tetraphosphate) can beenhanced by activation of the tributylamine salt (0.1 mmol) with a largeexcess of DCC (0.1 g, 0.5 mmol); in this case the depositeddicyclohexylurea was removed by filtration, the reaction mixtureextracted with ether (10 mL) and the residue dissolved in dry DMF priorto treatment with tributylamine UMP (0.2 mmol). Upon chromatographicseparation of the reaction mixture and quantitation by ultravioletabsorption as in Example 3A above, the uridine tetraphosphate productconstituted 50.7% of the uridylate species in the mixture, correspondingto a conversion from UTP of 95.9%.

EXAMPLE 4A Method for the Production of Diuridine Tetraphosphate UsingUridine 5′-Monophosphate Activated with Carbonyldiimidazole

[0054] Uridine 5′-monophosphate (UMP) (0.324 g, 1.0 mmol) was dissolvedin a mixture of dry DMF (5 mL) and tributylamine (237 μL, 1 mmol) thesolution was evaporated to dryness, then twice more with DMF to yieldthe anhydrous tributylamine salt. The residue was dissolved in DMF (5mL) and carbonyldiimidazole (CDI) (0.81 g, 5 mmol) added. The solutionwas set aside for 3 hours, then methanol 324 μL, 8 mmol) added todestroy the excess of CDI. The solution was set aside for one hour.Tributylamine pyrophosphate (Sigma, 0.228 g, 0.5 mmol) was added and thesuspension stirred under nitrogen at room temperature. After 3 hours thereaction was quenched with water and the mixture subjected to HPLC as inExample 3A above. Yield of P¹,P⁴-di(uridine 5′-tetraphosphate) asquantitated by its absorbance at 263 nm was 9.3%.

EXAMPLE 4B Method for the Production of Diuridine Tetraphosphate UsingUridine 5′-Monophosphate Activated with Diphenyl Phosphochloridate

[0055] The anhydrous tributylamine salt of UMP (1.0 mmol), preparedessentially as above, was dissolved in a mixture of dry dioxane (5 mL)and DMF (1 mL). Diphenyl phosphochloridate (0.3 mL) and tributylamine(0.3 mL) were added and the solution set aside at room temperature for 3hours. The solvent was evaporated and the residue shaken with ether (˜10mL), then set aside at 4° C. for 30 minutes. The ether was decanted andthe residue was dissolved in a solution of tributylamine pyrophosphate(0.228 g, 0.5 mmol) in DMF (3 mL). The solution was stored undernitrogen at room temperature. After 3 hours the reaction was quenchedwith water and the mixture subjected to HPLC as in Example 3A above.Yield of P¹,P⁴-di(uridine 5′-tetraphosphate) as quantified by itsabsorbance at 263 nm was 9.6%.

EXAMPLE 5 Method for the Production of Diuridine Tetraphosphate UsingUridine, Phosphorus Oxychloride and Pyrophosphate

[0056] Uridine (Aldrich, 0.244 g, 1 mmol) was dissolved in trimethylphosphate (Aldrich, 5 mL) and tributylamine (466 uL, 2 mmol) added. Thesolution was stirred at 0 degrees during the addition of phosphorusoxychloride (0.153 g (93.2 uL), 1 mmol), and the resulting suspensionstirred at 0° C. for 3 hours. Tributylamine pyrophosphate (0.228 g) wasadded and the suspension stirred at room temperature for 3 hours. Thereaction was quenched with 1.0 M aqueous triethylamine bicarbonate andthe mixture extracted with methylene chloride to remove trimethylphosphate. The aqueous solution was subjected to HPLC as in Example 3Aabove. Conversion of uridine to P¹,P⁴-di(uridine 5′-tetraphosphate) asquantitated by absorbance of the latter at 263 nm was 6.83%.

EXAMPLE 6 Method for the Production of Diuridine Tetraphosphate UsingUridine 5′-Monophosphate and Pyrophosphoryl Chloride

[0057] Uridine 5′-monophosphate (UMP) (64.8 mg, 0.2 mmol) was dissolvedin dry pyridine (1 mL) and stirred in ice during the addition ofpyrophosphoryl chloride (13.9 uL (25 mg), 0.1 mmol). The solution becamecloudy almost immediately, then a copious semicrystalline whiteprecipitate formed which became a gummy mass within 1-2 minutes. Themixture was stored at room temperature overnight, the quenched withwater and subjected to HPLC as in Example 3A above. Yield ofP¹,P⁴-di(uridine 5′-tetraphosphate) as quantitated by its absorbance at263 nm was 15.8%. A substantial amount of P¹,P³-di(uridine5′-triphosphate) (25.4%) was obtained as the major by-product.

EXAMPLE 7 Aqueous Stability and Solubility of P¹,P⁴-di(uridine5′-tetraphosphate), Tetrasodium salt

[0058] The solubility of P¹,P⁴-di(uridine 5′-tetraphosphate),tetrasodium salt in water was determined by adding portions of solid toa known volume of deionized water until the solution became turbid. Themaximum solubility in water was thus determined to be ca. 900 mg/mL.Stability studies of the solid or aqueous solutions incubated at low (5°C.) and elevated temperatures (40° C.) showed that less than 1.5%degradation occurs over a three month period as determined by HPLCanalysis. The tetrasodium salt of P¹,P⁴-di(uridine 5′-tetraphosphate)was thus determined to have an excellent solubility and stabilityprofile suitable for pharmaceutical applications.

EXAMPLE 8 Toxicity of P¹,P⁴-di(uridine 5′-tetraphosphate), TetrasodiumSalt in Animals

[0059] The nonclinical toxicologic profile of P¹,P⁴-di(uridine5′-tetraphosphate), tetrasodium salt has been evaluated in a battery ofgenetic toxicology assays that include the bacterial reverse mutationassay, the in vitro mammalian cytogenetic test, the in vitro mammaliancell gene mutation test, and the micronucleus cytogenetic assay in mice.A study in rabbits examined local ocular tolerance and subchronic oculartoxicity after multiple daily administrations over a six-week period. Inaddition, P¹,P⁴-di(uridine 5′-tetraphosphate), tetrasodium salt has alsobeen tested in two single-dose acute inhalation toxicity studies in ratand dog, and one single-dose acute intravenous toxicity study in dogs.

[0060] The results of these studies show that P¹,P⁴-di(uridine5′-tetraphosphate), tetrasodium salt is nongenotoxic in a battery ofgenetic toxicology assays. No adverse findings were seen in the oculartoxicology studies. A low degree of acute toxicity was seen in singledose inhalation (rats, dogs) and intravenous (dogs) toxicity studies.P¹,P⁴-di(uridine 5′-tetraphosphate), tetrasodium salt was thereforedetermined to have an excellent toxicology profile with a wide safetymargin for dosing in humans.

EXAMPLE 9 Safety and Efficacy of P¹,P⁴-di(uridine 5′-tetraphosphate),Tetrasodium Salt in Normal Human Volunteers

[0061] P¹,P⁴-di(uridine 5′-tetraphosphate), tetrasodium salt wasevaluated in a Phase I, double-blind, placebo-controlled, escalatingdose, safety and tolerability study in 75 normal healthy malevolunteers. Forty non-smokers and 35 smokers were evaluated in 5 dosingcohorts of 16 volunteers, comprised of 12 receiving a single aerosolizeddose of P¹,P⁴-di(uridine 5′-tetraphosphate), tetrasodium salt (20-400mg) and 4 receiving placebo (normal saline). No serious adverse eventswere reported. There were no significant changes in FEV₁, FVC, MMEF,clinical laboratory, 12-lead ECG, or urinalysis results in either theplacebo or active drug groups. In smokers, P¹,P⁴-di(uridine5′-tetraphosphate), tetrasodium salt produced a 2-fold to 7-folddose-dependent increase in the weight of sputum expectorated within 5minutes of dosing, and stimulation of sputum expectoration was sustainedover the next hour of sputum collection. The effect of P¹,P⁴-di(uridine5′-tetraphosphate), tetrasodium salt to induce the expectoration ofsputum in non-smokers was also observed. In conclusion, P¹,p⁴-di(uridine5′-tetraphosphate), tetrasodium salt is safe and well-tolerated innormal male subjects and is effective in stimulating the expectorationof sputum when compared to placebo.

What is claimed is:
 1. The compounds of Formula I:

wherein: X is selected from the group consisting of: Na, NH₄ and H,provided that all X groups are not H.
 2. P¹,P⁴-di(uridine5′-tetraphosphate), tetrasodium salt.
 3. P¹,P⁴-di(uridine5′-tetraphosphate), tetrammonium salt.
 4. A process for the synthesis ofcompounds of Formula I, and pharmaceutically acceptable salts thereof,said process comprising: a) dissolving uridine or uridine nucleotidecompounds of Formulas IIa-d in a polar, aprotic organic solvent and ahydrophobic amine; b) phosphorylating with a phosphorylating agent ofFormulas IVa-b and/or activating with an activating agent of FormulasIIIa-c; and c) purification by chromatography of the compounds ofFormula I, and pharmaceutically acceptable salts thereof;

wherein: X is selected from the group consisting of: Na, NH₄ and H,provided that all X groups are not H;

and salts thereof;

and salts thereof;

and salts thereof;

wherein R¹ and R₂ are independently selected from the group consistingof: C₁-C₈ alkyl, C₁-C₈ cycloalkyl, C₁-C₈ optionally substituted alkyl,C₁-C₈ optionally substituted cycloalkyl substituted with hydroxy andamino groups, aryl, and optionally substituted aryl substituted withhydroxy and amino groups;

wherein X is independently selected from the group consisting of:imidazole, tetrazole and halogen;

wherein X is halogen and R¹ and R₂ are each independently selected fromthe group consisting of: C₁-C₈ alkyl, C₁-C₈ cycloalkyl, C₁-C₈ optionallysubstituted alkyl substituted with hydroxy and amino groups, C₁-C₈optionally substituted alkoxy substituted with hydroxy and amino groups,C₁-C₈ optionally substituted cycloalkyl substitued with hydroxy andamino groups, aryl, alkoxy, optionally substituted aryl substituted withhydroxy or amino groups, optionally substituted alkoxy substituted withhydroxy and amino groups, and halogen;

wherein X is halogen;

wherein X is selected from the group consisting of: oxygen, hydroxy,halogen, and salts thereof.
 5. The process of claim 4 , wherein thecompounds of Formula IIIa are selected from the group consisting of:dicyclohexylcarbodiimide and1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
 6. Theprocess of claim 4 , wherein the compounds of Formula IIIb are selectedfrom the group consisting of: carbonyldiimidazole andcarbonylditriazole.
 7. The process of claim 4 , wherein the compound ofFormula IIIc is selected from the group consisting of:diphenylphosphorochloridate, phenyl phosphorodichloridate,phenylphosphonic dichloride and diphenylphosphinic chloride.
 8. Theprocess of claim 4 , wherein the compound of Formula IVa is phosphorusoxychloride.
 9. The process of claim 4 , wherein the compound of FormulaIVb is selected from the group consisting of: pyrophosphoryl chlorideand pyrophosphate.
 10. The process for the preparation of compounds ofFormula I as described in claim 1 , and pharmaceutically acceptablesalts thereof, said process comprising dissolving a compound of FormulaIIc as described in claim 4 in a polar, aprotic organic solvent and ahydrophobic amine, treating with an activating agent from FormulasIIIa-c as described in claim 4 , and subsequent purification bychromatography.
 11. A process for the preparation of compounds ofFormula I as described in claim 1 , and pharmaceutically acceptablesalts thereof, said process comprising dissolving a compound of FormulaIIb as described in claim 4 in a polar, aprotic organic solvent and ahydrophobic amine, treating with an activating agent from FormulasIIIa-c as described in claim 4 , followed by reacting with a compound ofFormula lId as described in claim 4 , and subsequent purification bychromatography.
 12. A process for the preparation of compounds ofFormula I as described in claim 1 , and pharmaceutically acceptablesalts thereof, said process comprising dissolving a compound of FormulaIId as described in claim 4 in a polar, aprotic organic solvent and ahydrophobic amine, treating with an equimolar amount of activating agentfrom Formulas IIIa-c as described in claim 4 , followed by reacting witha compound of Formula IIb as described in claim 4 , and subsequentlypurification by chromatography.
 13. A process according to claim 12whereby an excess of activating agent is used.
 14. A process for thepreparation of compounds of Formula I as described in claim 1 , andpharmaceutically acceptable salts thereof, said process comprisingdissolving a compound of Formula IIb as described in claim 4 in a polar,aprotic organic solvent and a hydrophobic amine, treating with anactivating agent from Formulas IIIa-c as described in claim 4 , followedby reacting with a compound of Formula IVb as described in claim 4 , andsubsequent purification by chromatography.
 15. A process for thepreparation of compounds of Formula I as described in claim 1 , andpharmaceutically acceptable salts thereof, said process comprisingdissolving a compound of Formula IIa as described in claim 4 in a polar,aprotic organic solvent and a hydrophobic amine, treating withphosphorylating agents from Formulas IVa-b as described in claim 4 , andsubsequent purification by chromatography.
 16. A process for thepreparation of compounds of Formula I as described in claim 1 , andpharmaceutically acceptable salts thereof, said process comprisingdissolving a compound of Formula IIb as described in claim 4 in a polar,aprotic organic solvent and a hydrophobic amine, treating with aphosphorylating agent from Formula IVb as described in claim 4 , andsubsequent purification by chromatography.
 17. A method of treatingchronic obstructive pulmonary diseases in a mammal by administering aneffective chronic obstructive pulmonary disease treatment amount of acompound of Formula I as described in claim 1 .
 18. A method of treatingsinusitis, otitis media or nasolacrimal duct obstruction in a mammal byadministering an effective mucus secretion clearing amount of a compoundof Formula I as described in claim 1 .
 19. A method of treating dry eyein a mammal by administering an effective dry eye treatment amount of acompound of Formula I as described in claim 1 .
 20. A method of treatingretinal detachment in a mammal by administering an effective retinaldetachment treatment amount of a compound of Formula I as described inclaim 1 .
 21. A method of treating or preventing pneumonia in a mammalby administering an effective pneumonia prevention treatment amount of acompound of Formula I as described in claim 1 .
 22. A method offacilitating sputum induction in a mammal by administering an amount ofa compound of Formula I as described in claim 1 effective to facilitatesputum induction.
 23. A method of facilitating expectoration in a mammalby administering an amount of a compound of Formula I as described inclaim 1 effective to facilitate expectoration.
 24. A method of treatingfemale infertility or vaginal irritation due to vaginal dryness byadministering an effective vaginal dryness treatment amount of acompound of Formula I as described in claim 1 .
 25. A pharmaceuticalcomposition comprising a compound of Formula I as described in claim 1together with a pharmaceutically acceptable carrier therefor.